There is good evidence that many of the interactions between blood cells and endothelial cells lining microvessels. which play an important role in such pathological processes as ischemia-reperfusion injury, involve signaling mechanisms in which the regulation of cellular ion fluxes and cell volume play a major role. This project focuses primarily on basic mechanisms of regulation of anion content and volume, in both neutrophils and endothelial cells, with an emphasis on using the unique opportunities of this program project to relate this cellular regulatory processes to functional studies of their effects on the behavior of cells in microcirculatory model systems. The first aim is directed toward understanding the mechanism of and effects of the initial decrease and later increase of neutrophil C1 content ("C1 pulse"), which is a common feature of activation by various agonists and where there is abundant pharmacological evidence for functional importance. A combination of isotope marker, chemical probe, and electrophysiological techniques will be used to determine the mechanisms operative in each phase of this process. This knowledge will be used in collaborative studies with other projects to assess the functional importance of the C1 pulse in affecting cell mechanical properties, granule release, display of surface adhesion molecules, and migration through endothelial cell layers. The second aim involves analysis of the way in which volume regulatory processes in endothelial cells may contribute to pathological events in hemorrhagic shock and ischemia, and on how these proteins could affect interaction with circulating neutrophils. Research will focus on the mechanism of endothelial cell swelling under ischemic and acidic conditions, and the reason for failure of volume-regulatory mechanisms, as well as alterations of neutrophil interactions with endothelial cells under theses conditions. Also, the effects of shear stress on endothelial cell volume and on ion regulation will be examined, under conditions where endothelial cells are grown on surfaces with various curvatures. These studies provide a basis for understanding changes in endothelial cell function under conditions of circulatory stress in terms of fundamental alterations of cell regulatory mechanisms.